Beta turn peptidomimetic cyclic compounds

ABSTRACT

Proteolytically stable small molecule β-turn peptidomimetic compounds have been identified as agonists or antagonists of neurotrophin receptors, such as TrkA. A compound of particular interest binds the immunoglobulin-like C2 region of the extracellular domain of TrkA, competes the binding of another TrkA ligand, affords selective trophic protection to TrkA-expressing cell lines and neuronal primary cultures, and induces the differentiation of primary neuronal cultures. The small β-turn peptidomimetic compounds of the invention can activate a tyrosine kinase neurotrophin receptor that normally binds a relatively large protein ligand. Such compounds that bind the extracellular domain of Trk receptors are useful pharmacological agents to address disorders where Trk receptors play a role, by targeting populations selectively.

TECHNICAL FIELD

[0001] This invention relates to β-turn peptidomimetic cyclic compoundsuseful in the field of neurotrophin receptor agonists and antagonists;the invention also relates to neurotrophin receptor agonist andantagonist pharmaceutical compositions; a method of treating orpreventing disorders mediated or regulated by neurotrophin receptors;use of β-turn peptidomimetic cyclic compounds in evaluating structuralrequirements of neurotrophin receptor agonists and antagonists;identification of receptor subdomains desired for ligand docking, andnovel β-turn peptidomimetic cyclic compounds.

BACKGROUND ART

[0002] Tyrosine kinase A (TrkA) is a transmembrane tyrosine kinasereceptor with high selectivity for the neurotrophin nerve growth factor(NGF). Related neurotrophins include Brain Derived Neurotrophic Factor(BDNF) which binds tyrosine kinase B (TrkB) receptors, andNeurotrophin-3 (NT-3) which prefers binding to tyrosine kinase C (TrkC)receptors.

[0003] Docking of TrkA with NGF initiates receptor dimerization,catalytic phosphorylation of cytoplasmic tyrosine residues on thereceptor, and a cascade of cell signaling events. These signals lead toprevention of apoptotic cell death, to promotion of cellulardifferentiation and axon elongation, and upregulation of choline acetyltransferase (ChAT). The same applies to other neurotrophins, except thatdifferent cell populations respond selectively based on their receptorexpression patterns. NGF will be used as the proof of principle but thenotions apply to all neurotrophins (NTFs).

[0004] Several neuronal cell types that are implicated in importantdisease states express TrkA and therefore respond to NGF, includingsensory, sympathetic and cholinergic neurons. It has been suggested thatNGF therapy may delay the onset of Alzheimer's disease preventfunctional loss associated with cerebral stroke, and ameliorateperipheral diabetic neuropathies. Other applications proposed for NGFinclude treatment of neuronal damage, and targeting ofneuroectoderm-derived tumors. For a review of disease targets see(Saragovi and Burgess, 1999).

[0005] Despite the therapeutic potential of NGF clinical trialsfeaturing this protein have been disappointing (Saragovi and Burgess,1999). There are several reasons for this: inherent drawbacks associatedwith the use of polypeptides applied as drugs, in vivo instability, andpleiotrophic effects due to activation of signals that were notintentionally targeted. Moreover, the NGF protein is relativelyexpensive to produce for medicinal applications.

[0006] Agonists of TrkA, TrkB and Trk C and p75 receptor would haveutility in the treatment and prevention of tyrosine kinase receptormediated disorders, for example, chronic or acute neurodegeneration,pain, cancer, cerebral stroke, neuromas, ocular nerve diseases, such asglaucoma, and Alzheimer's disease.

[0007] Strategies that result in agonists of tyrosine kinase receptorshave not been well established. Previously, ligand mimicry and antibodymimicry strategies have been attempt to generate peptide analogs of twoagonists directed to the extracellular domain of TrkA: the naturalligand NGF; (LeSauteur et al, 1995), and monoclonal antibody (mAb) 5C3(LeSauteur et al., 1996). TrkA binding is mediated by discrete β-turnregions of these ligands. Only certain cyclic β-turn analogs were active(Beglova et al., 1998), and other conformers or linear peptides wereinactive.

[0008] Disclosure

[0009] This invention seeks to provide a neurotrophin receptor agonistor antagonist pharmaceutical composition.

[0010] The invention also seeks to provide a method of treating orpreventing disorders of tissues where neurotrophin receptors pay a role.

[0011] Still further this invention seeks to provide β-turnpeptidomimetic cyclic compounds for use in evaluating structuralrequirements of neurotrophin receptor agonists and antagonists.

[0012] The invention also seeks to provide a novel class of β-turnpeptidomimetic cyclic compounds.

[0013] In accordance with one aspect of the invention there is provideda neurotrophin receptor agonist or antagonist pharmaceutical compositioncomprising an acceptable neurotrophin receptor agonistic or antagonisticamount of a neurotrophin mimicking β-turn peptidomimetic cycliccompound, in association with a pharmaceutically acceptable carrier.

[0014] In accordance with another aspect of the invention there isprovided a method of treating or preventing a neurotrophin receptormediated or regulated disorder in a patient comprising administering toa patient in need, an acceptable neurotrophin receptor agonistic orantagonistic amount of a neurotrophin mimicking β-turn peptidomimeticcyclic compound.

[0015] In accordance with still another aspect of the invention there isprovided use of β-turn peptidomimetic cyclic compounds in evaluatingstructural requirements of neurotrophin mimicking β-turn peptidomimeticcyclic compounds.

[0016] In accordance with yet another aspect of the invention there isprovided a β-turn peptidomimetic cyclic compound of formula (I)

[0017] The compounds of formula (I) include the material termed D3 andderivatives thereof.

[0018] Some of these derivatives may be simple and obvious modificationslike biotinylated forms and molecules wherein two such units are linkedby dimers. Other obvious derivatives of D3 include the following. Theside chains R¹-R⁶ could include any alkyl or aryl substituent found innatural and unnatural amino acids.

[0019] The side chains typical of the protein amino acids (eg Arg, Trp,His) are of particular interest, and many compounds in this series havebeen prepared herein, but the diversity of structures that are easilygenerated derivatives of D3 include many types of functional groups. Theconstituent amino acids may be N-alkyl, N-aryl, α,α-dialkyl, and cyclicderivatives such as might be formed from cyclopropane amino acids.

[0020] The substituent(s) Y may be hydrogen or one or two aromaticsubstituents for example nitro, amino, halo, alkyl for example alkyl of1 to 6, preferably 1 to 4 carbon atoms, and aryl for example phenyl ornaphthyl. The alkyl and aryl substituents Y may be unsubstituted orsubstituted, suitable substituents being nitro and alkyl of 1 to 6carbon atoms.

[0021] Y may also be derivatized with a functional group, for examplebiotin. The group X may be any nucleophilic atom like O, N, S, P, Se,but also others such as C, or may be an alkylene radical typically of 1to 6 carbon atoms, for example methylene; or NH. The point of connectioncould be ortho- or meta- to the benzoyl carbonyl. Permissible values of“n” are 0, 1, 2, 3, 4, and 5. The liking side chain that incorporates Xmay be aliphatic as indicated in structure (I) aromatic orheteroaromatic.

[0022] The side chain alkyl groups R¹, R², R³, R⁴, R⁵, and R⁶ can bevaried in many ways to enhance the biological activities of thesematerials. Typically R¹, R², R³, and R⁴ are amino acid side-chainsubstituents found in the twenty protein-amino acids or side-chains verysimilar to these, for example the side-chains of glutamic acid, lysine,ornithine and threonine, in either enantiomeric configuration. If the R¹substituent is an amino acids side chain, the other substituent on thatcarbon, R², will typically be hydrogen, but could also be methyl, ethylor benzyl. Alternatively, R¹ and R² could be joined as in cyclopropane,cyclobutane, cyclopentane, and cyclohexane, residues. R³ and R⁴ arerelated in the same way as R¹ and R² as described above. That is, one ofthem will be an amino acid side chain or something very similar to this.The other of these two substituents is hydrogen in most cases, but couldalso be methyl, ethyl, propyl, benzyl or some simple alkyl system asdescribed above.

[0023] There is much scope for variation in R⁵ and R⁶ but by far themost common substituent at these positions is hydrogen. Thosesubstituents might also be designed to correspond to one of the sidechains of the twenty protein-amino acids, notably methyl.

[0024] The compounds (I) are more especially compounds prepared from thetwenty protein amino acids or simple analogs of these, including theirenantiomers, N-alkyl, N-aryl, α,α-dialkyl, and cyclic amino acids. Sidechains found to be particularly conducive to biological activities areR¹ and R³ as side chains of lysine, glutamic acid, tyrosine,iso-leucine, asparagine, and threonine, R², R⁴, R⁵, and R⁶ as hydrogen.One or more of the side chain are selected especially to correspond toside chains within turn regions of the neurotrophin proteins that thecyclic compound mimics, eg NGF, NT-3, NT 4/5 and/or BDNF.

[0025] In general the macrocyclic compounds have 13 to 16 membered ringswhere the X substituent is O, N, S, SO, or SO₂. The molecular fragmentsZ and Y are typically aromatic rings based on a simple ring system,particularly substituted benzenes. Nitro, amino, chloro, bromo, andfluoro-substituted benzenes are all permissible at this position.

[0026] Overall, several hundreds of compounds have been prepared thatconform to the structure given above. Some specific examples ofcompounds (I) and which have been tested in assays forneurotrophin-related activities are listed below.

[0027] Examples of embodiments that mimic neurotrophic activity areagents termed D3, P27, D53b-d, 25, P56, P57, P58, P39, D21, D46, D40,and P23. Examples of embodiments that antagonize neurotrophic activityare P42 and P43. These agents are shown below:

DETAILED DESCRIPTION OF INVENTION

[0028] Small, proteolytically stable molecules with neurotrophicactivity, selective for cells expressing neurotrophin receptors (Trktyrosine kinase receptors, and p75 receptors) have been developed in thepresent invention.

[0029] Based on the pharmacophores of the mAb 5C3 and of NGF peptideanalogs described previously, a focussed library of β-turnpeptidomimetic compounds has been synthesized.

[0030] This library of compounds is composed of β-turn peptidomimeticcyclic compounds. These compounds, in particular, mimic neurotrophins,and thus are agonists or antagonists for the neurotrophin receptors, orcan be employed in the screening and/or evaluation of necessarystructural requirements of such agonists and antagonists.

[0031] In general the cyclic compounds have a macrocyclic ring of 13 to17, more especially 14, 15 or 16 ring atoms; and the ring is formedpredominantly by a carbon and nitrogen backbone having side chains ofamino acids which may be natural or synthetic.

[0032] The ring may be characterized by one or more side chains on thepeptido linkage, especially at the i, i+1, i+2 and i+3 positions. Thecyclic compound typically has 1, 2 or 3 side chains.

[0033] The one or more side chains more especially correspond to sidechains within. β-turns of a neurotrophin protein which the cycliccompound mimics, and in particular the β-turns correspond to the β-turnsof a neurotrophin as NGF, NT-3, NT 4/5 and BDNF.

[0034] The β-turn peptidomimetic cyclic compounds of the invention, may,in particular embodiments be represented by the formula (I), as definedhereinbefore.

[0035] In formula (I), the macrocyclic ring containing R₁ to R₆ suitablyhas 13 to 17 and preferably 14, 15 or 16 ring atoms.

[0036] In preferred embodiments X is O, S or NH and R1, R3, R5 and R6are hydrogen atoms. When Y is a substituent it may function as a labelor precursor of a label, which label can be employed in the assessmentof the direct binding, the in vivo distribution, and agonist orantagonist capability of the cyclic compound.

[0037] The LINKER group functions as a linking group to form dimers ofthe compound (I) by reaction with a homo bifunctional compound such aspolyethylene glycol. Suitable LINKER groups include NH₂, OH, SH, COOH,CH₃CO and CHO.

[0038] Representative compounds of formula (I) which have been producedas part of the afore-mentioned library are indicated hereinafter.

[0039] The cyclic compounds of the invention may be prepared accordingto the procedure described by Feng et al in J. Am. Chem. Soc. 1998, 120,10768-1076. The general type of reaction is illustrated hereinafter

[0040] This is a solid-phase S_(N)AR macrocyclization in which aminoacids sequentially form peptide linkages of the macrocyclic ring. Ifnecessary side chains on the peptido linkage may be protected, forexample, as t.butyl esters or BOC-amides.

[0041] The substituent Y in formula (I) may be, for example, NO₂ whichcan be readily reduced to amino which may be employed to develop abiotin label; other labels that could be attached to substituent Yinclude radioactive elements such as iodine and technetium.

[0042] The invention is illustrated hereinafter by reference to thecompound EKHse-G-OH referred to hereinafter for convenience as D3 andits biotin derivative referred to hereinafter as D3-Biotin and thecompound EKCys referred to hereinafter for convenience as C59 and whichis an analog of D3, the structures being indicated hereinafter

[0043] The neurotrophin receptor agonist or antagonist properties aremost effectively utilized in the treatment of neurotrophin receptormediated disorders when the cyclic compound is formulated into novelpharmaceutical compositions with a pharmaceutically acceptable carrieraccording to conventional pharmaceutical compounding techniques.

[0044] The novel compositions contain at least a therapeuticneurotrophin receptor agonist or antagonist amount of the active cycliccompound. Generally, the composition contains up to 0.1 to 100 mg/kgbody weight of the patient of the cyclic compound. Concentratecompositions suitable for dilutions prior to use may contain 90 percentor more by weight. The compositions include compositions suitable fororal, rectal, topical, parenteral (including subcutaneous,intramuscular, and intravenous), pulmonary (nasal or buccal inhalation),nasal administration, or insufflation. The compositions may be prepackedby intimately mixing the cyclic compound with the components suitablefor the medium desired.

[0045] When oral administration is to be employed, it may be with aliquid composition. For liquid preparations, the therapeutic agent isformulated with liquid carriers such as water, glycols, oils, alcohols,and the like, and for solid preparations such as capsules and tablets,solid carriers such as starches, sugars, kaolin, ethyl cellulose,calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose,generally with lubricant such as calcium stearate, together withbinders, disintegrating agents and the like. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage form. It is especially advantageous to formulate thecompositions in unit dosage form (as hereinafter defined) for ease ofadministration and uniformity of dosage. Composition in unit dosage formconstitutes an aspect of the present invention.

[0046] The cyclic compound also may be formulated in therapeuticcompositions for intravenous or intraperitoneal injection and may bepresented in unit dosage form in ampoules or in multidose containers, ifnecessary with an added preservative. The compositions may also takesuch forms as suspensions, solutions or emulsions in oily or aqueousvehicles such as sodium chloride or dextrose in water, and may containformulating agents such as suspending, stabilizing and/or dispersingagents. Buffering agents as well as additives such as saline or glucosemay be added to make the solutions isotonic. The cyclic compound alsomay be solubilized in alcohol/propylene glycol or polyethyleneglycol fordrip intravenous administration. Alternatively, the active ingredientsmay be in powder form for reconstituting with a suitable vehicle priorto administration.

[0047] The term “unit dosage form” herein refers to physically discreteunits, each unit containing a predetermined quantity of activeingredient calculated to produce the desired therapeutic effect inassociation with the pharmaceutical carrier. Examples of such unitdosage forms are tablets, capsules, pills, powder packets, wafers,measured units in ampoules or in multidose containers and the like. Aunit dosage of the present invention will generally contain from 100 to200 milligrams of one of the cyclic compounds.

[0048] For administration by inhalation, the cyclic compounds of thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs of nebulisers. The cycliccompounds may also be delivered as powders which may be formulated andthe powder composition may be inhaled with the aid of an insufflationpowder inhaler device. The preferred delivery system for inhalation is ametered dose inhalation (MDI) aerosol, which may be formulated as asuspension or solution of the cyclic compound in suitable propellants,such as fluorocarbons or hydrocarbons.

[0049] Another method of administration is insufflation, particularly ifthe infection has spread to the ears and other body cavities.

[0050] If the application is to be topical, the cyclic compound may beformulated in conventional creams and ointments such as whitepetrolatum, anhydrous lanolin, cetyl alcohol, cold cream, glycerylmonostearate, rose water and the like.

BRIEF DESCRIPTION OF DRAWINGS

[0051]FIG. 1 demonstrates that D3 induces the partial differentiation ofembryonic DRG cultures; when applied alone; and synergistic enhancementof the effect of suboptimal NGF concentrations.

[0052]FIG. 2 demonstrates that D3 enhances cell surface TrkA.TrkAhomodimers;

[0053]FIG. 3 illustrates NGF with highlighted turn regions believed tobe critical for binding to the TrkA receptor (two zinc atoms are presentin the dimer but are omitted for overall clarity; and

[0054]FIG. 4 illustrates the structure of NG-3/BDNF heterodimer withcorresponding turn regions of NT-3 highlighted.

DETAILED DESCRIPTION OF DRAWINGS

[0055] In FIG. 1 primary neuronal DRG cultures were treated as indicatedfor 8 days, and cell differentiation was studied morphometrically.Magnification 60×. Pictures representative of 3 independent experiments.

[0056] In FIG. 2, 4-3.6 cells were exposed to TrkA ligands as per Table5 (lanes 1-4) or no ligand (lanes 5 and 6), and chemically cross-linked(lanes 1-5) or not cross-linked (lane 6). Cell lysates were westernblotted with anti-TrkA 203 antisera. The intensity of the M_(r) 300 kDaband was analyzed densitometrically from 4 experiments standardized to 1nM NGF.

[0057] The compound D3 is a small, selective, and proteolytically stableagonist of the TrkA receptor. Furthermore, the docking site of D3 ontoTrkA has been evaluated and demonstrates that a small peptidomimeticligand can agonize a tyrosine kinase neurotrophin receptor that normallybinds a relatively large protein ligand. The compounds of the inventionthus offer an alternative therapeutic strategy with pharmacologicalagents that selectively target neuronal populations expressing specificneurotrophin receptors on the cell surface.

EXAMPLES

[0058] Materials and Methods

[0059] Preparation of D3 and D3-biotin.

[0060] Compound D3 was prepared according to methods previously outlinedfor related compounds (Feng et al., 1998). FMOC-Gly, FMOC-Hse(Trt),FMOC-Lys(BOC), FMOC-Glu(OtBu), then 2-fluoro-5-nitrobenzoyl chloridewere coupled (di-iso-propylcarbodiimide activation, 20% piperidine inDMF to remove FMOC groups) to TentaGel S PHB resin at 0.18 mmol/gloading. The supported peptide was treated six times with 1% TFA/4 5HSiiPr₃ in CH₂Cl₂ for 5 min to remove only the Trt-protection.Cyclization was effected by treatment with 5.0 equivalents of K₂CO₃ inDMF for 40 h. After 90% TFA/5% H₂O/5% HSiiPr₃ cleavage, the finalproduct was purified by reverse phase HPLC. D3 and its derivatives weresoluble in water to 5 mg/ml (the highest concentration tested).

[0061] D3-biotin was prepared in the same way as D3, except that afterthe cyclization the nitro group was reduced by treatment with 10equivalents of SnCl₂.2H₂O in DMF for 20 h. After reduction, FMOC-Gly,then biotin-N-hydroxysuccinimide was coupled to the newly formedarylamine. The product was then cleaved from the resin. The finalproduct was purified by reverse phase HPLC.

[0062] Cell lines: B104 rat neuroblastomas express p75 receptors but donot express any of the Trks (TrkA− p75+). The 4-3.6 cells are B104 cellsstably transfected with human trkA cDNA, and express equal levels of p75and TrkA (TrkA+ p75+). For screening agents that activate or antagonizeTrkC receptors, NIH3T3 fibroblasts were stably transfected with humanTrkC cDNA. These cells respond to the ligand NT-3. For screening agentsthat activate or antagonize TrkA receptors, NIH3T3 fibroblasts werestably transfected with human TrkA cDNA. These cells respond to theligand NGF. Wild type NIH3T3 fibroblasts were used as controls becausethey do not respond to any neurotrophin ligand.

[0063] Generation of Human TrkA-Rat TrkB Chimeras in HEK293 Cells

[0064] The IgG-C2 domain of human TrkA was generated using uniquerestriction sites in the primers to allow exchange with thecorresponding rat TrkB domain. The chimeric receptors were constructedby subcloning the human TrkA IgG-C2 domain into the correspondingrestriction sites of the rat trkB cDNA reported in a previous work(Perez et al., 1995). Chimeric constructs were confirmed by sequencing,and were cloned into the pCDNA3 expression vector that contains aselection gene providing resistance to neomycin (G418, GIBCO). HEK293cells were transfected using the lipofectamine plus method (GIBCO),selected with neomycin (0.5 mg/ml) and at least 3 independent subcloneswere obtained by limiting dilution techniques (293-TrkB/A-IgC2 chimera).Western blot analysis with polyclonal antibody 203 directed to the Trkintracellular domain and cell surface FACScan analysis with polyclonalantibody directed to the TrkA extracellular domain indicated that allstable subclones express comparable levels of chimeric receptors.

[0065] Dissociated Neuronal Dorsal Root Ganglia Cultures:

[0066] Fetal rat DRG primary cultures were established from SpragueDawley day 17 rat embryos. All ganglia were dissected and dissociatedfirst enzymatically with trypsin and then mechanically. Dissociatedcells were cultured (10⁵ cells/well) in 96 well plates pre-coated withcollagen, and grown for a total of 8 days in Neuro Basal Mediumcontaining N2 supplement (GIBCO, Toronto), antibiotics, and L-glutamine.These DRG cultures are ˜85% TrkA-expressing and are heavily dependent onTrkA signals for survival.

[0067] Septal Neuronal Cultures:

[0068] Cell cultures were established from the septal area of 17-day-oldrat embryos. In brief, tissue was incubated in PBS containing trypsinand DNase. Tissue pieces then were mechanically dissociated. Aftercentrifugation, the pellet was suspended in Leibovitz's L-15 medium.Cells were plated onto 96-multiwell NUNC dishes (10⁵ cells/well) coatedwith poly-D-lysine (5 μg/ml). Pure cultures of septal neurons weretreated 1 day after plating. Drugs, prepared in medium, were addeddirectly to the cells without changing the initial medium. Theincubation continued for 8 days, at which time ChAT activity wasevaluated.

[0069] D3•TrkA Binding Assays.

[0070] Direct binding studies: were done as described (Saragovi et al.,1998) using 6 ng/well of recombinant baculovirus TrkA-extracellulardomain protein (TrkA-ECD) or control bovine serum albumin (BSA, FractionV, Boehringer Mannheim) immobilized onto 96-well microtest plates. Wellswere blocked with binding buffer (BB: PBS with 1% BSA) for 1 hour. Then,50 ng/well of biotinylated D3 were added as primary reagent in BB for 40min in the absence or presence of excess non-biotinylated D3 ascompetitor. Wells were washed 5 times with BB, and horseradishperoxidase (HRP)-coupled avidin (Sigma) was added as secondary reagentfor 30 min. Plates were washed in BB, and peroxidase activity wasdetermined colorimetrically using 2,2-azinobis (3-ethylbenzthiazolinesulfonic acid) (ABTS, Sigma). The optical density (OD) was measured at414 nm in a Microplate reader (Bio-Rad). Assays were repeated at leastthree times, n=4.

[0071] FACScan binding assays: 4-3.6 cells (2×10⁵) in FACScan bindingbuffer (PBS, 0.5% BSA, and 0.1% NaN3) were immunostained as described(Saragovi et al., 1998). Saturating anti-TrkA mAb 5C3, or anti-p75 mAbMC192, or control non-binding IgGs were added to cells for 1 hour at 4°C., in the presence or absence of D3 as competitor. Excess primaryantibody was washed off, and cells were immunostained withfluorescinated goat-anti-mouse IgG secondary antibody. Cells wereacquired on a FACScan and mean channel fluorescence of bell-shapedhistograms were analyzed using the LYSIS II program.

[0072] Binding Competition: studies were as described for direct bindingassays to TrkA-ECD, except that as primary reagent 50 ng anti-TrkA mAb5C3/well were added in BB, in the presence or absence of D3 or controlsas competitors as described (Saragovi et al., 1998). Wells were washed 5times with BB, and HRP-coupled goat anti-mouse was added as secondaryreagent for 30 min. Plates were washed in BB, and peroxidase activitywere determined. Assays were repeated at least three times, n=4.

[0073] Cell Survival Assays.

[0074] Primary DRG cultures: After a total of 8 days of culture with theindicated test or control ligands, cell survival were studied using the3(4,5-Dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide calorimetric(MTT) assay, and by microscopic observation.

[0075] Cell lines: 5,000 cells/well in protein-free media (PFHM-II,GIBCO, Toronto) containing 0.2% bovine serum albumin (BSA) (Crystallinefraction V, Sigma, St. Louis, Mo.) were seeded in 96 well plates(Falcon, Mississauga, Ontario). The cultures were untreated, or treatedwith the indicated test or control ligands. Cell viability wasquantitated using the MTT assay after 56-72 hours of culture. Percentprotection was standardized from optical density (OD) readings relativeto optimal NGF (1 nM)=100%. The OD of untreated cells were subtracted.The higher optical density of untreated primary cultures is likely dueto cellular heterogeneity and to endogenous production of limitingamounts of growth factors.

[0076] Measurement of CHAT Activity.

[0077] At day 8 of culture, the medium was aspirated, and ice-cold lysisbuffer (10 mM sodium phosphate, pH 7.4/0.1% TritonX-100—Trade-mark) wasadded. ChAT activity assays were performed directly in the wells usingFonnum's method (Fonnum, 1975).

[0078] Detection of Putative TrkA-TrkA Homodimers.

[0079] Live 4-3.6 cells suspended in PBS were treated with the indicatedligand(s) for 40 min at 4° C. to allow binding. Cells were then washedin PBS, cross-linked with the membrane impermeable cross-linkerdisuccinimidyl suberate (DSS, Pierce; 1 mM DSS, 15 minutes at 15° C.).Unreacted DSS was quenched with 5 mM ammonium acetate. Then cells wereeither lysed directly in SDS sample buffer (whole cell lysate), or lysedin non-ionic detergent NP-40 and immunoprecipitated with anti-Trk oranti-p75 antibodies as described (LeSauteur et al., 1996). Similarresults were obtained with either method. For western blot analysis,equal amounts of protein or cell equivalents for each sample wereresolved in a 5-10% SDS-PAGE gradient, transferred to nitrocellulosemembranes (Xymotech Biosystems, Montréal, Qc), and blotted with anti-Trkpolyclonal antibody 203 that recognizes the intracellular domain of Trk.Blots were visualized using the enhanced chemiluminescence (ECL) system(New England Nuclear, Boston, Mass.).

Results

[0080] Synthesis of Focussed β-Turn Peptidomimetic Libraries

[0081] A solid phase synthesis was developed to yield a macrocyclic ringwith the i+1 and i+2 residues of a β-turn in the appropriateconformation. Approximately 60 compounds of this type were prepared(Feng et al., 1998), with amino acid side chains incorporated tocorrespond to β-turns of NGF and mAb 5C3 implicated in docking to TrkA(LeSauteur et al., 1996; LeSauteur et al., 1995). TrkA binding ismediated by discrete β-turn regions of these ligands. Cyclic peptideβ-turn analogs of NGF and of mAb 5C3 were active only in the appropriateconformation (Beglova et al., 1998).

[0082] C59 found to be inactive was used as a negative control. Abiotinylated form of D3, D3-biotin, was synthesized to carry out directbinding studies to TrkA. All ligands were highly soluble inphysiological buffers and did not require organic solvents.

[0083] D3 is a Selective Ligand of TrkA

[0084] FACScan analysis featuring the secondary fluorescent agentavidin-FITC was used to detect binding of D3-biotin to the cell surface(Table 1). The 4-3.6 cells (p75+TrkA+) had fluorescence approximately 4times greater for D3-biotin than for a background controlpeptide-biotin. Moreover, a 10-fold molar excess of D3 abolished bindingof D3-biotin. In contrast, no specific binding was measured for B104cells (p75+TrkA−). Since 4-3.6 cells are B104 cells stably transfectedwith TrkA cDNA and these cell lines are otherwise identical, the dataindicate that D3-biotin and D3 bind cell surface TrkA.

[0085] Similar binding data for D3-biotin was obtained by ELISA usingpure soluble TrkA extracellular domain (TrkA-ECD) produced inbaculovirus (see Table 3). These data further indicate that D3 binds tothe extracellular domain of TrkA, and that membrane lipids are notrequired.

[0086] D3 Binds Within an Agonistic Site of TrkA

[0087] Previously, mAb 5C3 was shown to act as a fall TrkA agonist. MAb5C3 binds with K_(d) 2 nM (LeSauteur et al., 1996) at an epitope withinthe IgC2 domain of TrkA near the NGF binding site. This site ispostulated to define a receptor “hot spot”. D3 and mAb 5C3 were testedto determine if they bind to overlapping receptor sites.

[0088] Two related assays tested the ability of D3 to compete for thebinding of the full TrkA agonist mAb 5C3. In the first test, aFACScan-based assay using intact cells, D3-induced a dose-dependentcompetitive decrease of mAb 5C3-TrkA interactions (Table 2, rows 2-5).On average, D3 exhibited an IC₅₀ of 4 μM. From experimental conditions aK_(d) ˜2 M for D3•TrkA interactions is estimated. Blocking of 5C3•TrkAinteractions by D3 is selective because the binding of mAb MC192directed to the p75 NGF receptor subunit was not blocked (Table 2, rows7 vs 8). Furthermore, inactive control C59 peptidomimetic did notinhibit the binding of either mAb 5C3 (Table 2, row 6) or mAb MC192.

[0089] The second test used purified recombinant TrkA extracellulardomain (TrkA-ECD) immobilized onto ELISA plates to assay competitiveblocking of 5C3•TrkA-ECD by D3. D3 exhibited a dose-dependent inhibitionof 5C3•TrkA-ECD interactions, but control inactive C59 peptidomimetichad no effect (Table 3). Since a K_(d) ˜2 nM was measured for 5C3•TrkAinteractions, from the experimental IC₅₀ a K_(d) ˜2 μM was calculatedfor D3•TrkA-ECD interactions. This calculation is consistent with thedata shown in Table 2. Interestingly, similar ELISA and RIA bindingassays revealed that D3 did not substantially block NGF•TrkA-ECDinteractions.

[0090] D3 Affords Trophic Activity Selectively via TrkA and isProteolytically Stable

[0091] Since D3 binds at or near an agonistic site of TrkA, trophiceffects were probed in cell survival assays using the quantitative MTTmethod. Several doses of D3 were tested. However, for clarity only nearoptimal concentrations are shown, which approximate the estimated K_(d).

[0092] Dissociated primary neuronal cultures from fetal dorsal rootganglia (DRG) are dependent on TrkA agonists for survival. Exogenous NGFshowed a dose-dependent trophic effect (Table 4, rows 24). D3 alone hada significant protective effect on DRG cultures (Table 4, row 5) butcontrol C59 did not (Table 4, row 6). Primary cultures are heterogeneousand low levels of neurotrophins are made endogenously, which explains arelatively high optical density for untreated cultures (Table 4, row 1).

[0093] Since D3 does not block NGF binding, potential synergy betweenNGF and D3 was assessed. D3 combined with different concentrations ofexogenous NGF demonstrated an additive or potentiating effect on DRGsurvival (Table 4, rows 7-9).

[0094] Similar results were obtained with other neuronal cell lines,wherein D3 potentiated the effect of low NGF concentrations (Table 5).Optimal protection of 4-3.6 cells (p75+TrkA+) and HEK293-TrkB/A-IgC2chimeras corresponded to treatment with 1 nM NGF (Table 5, row 2)whereas 10 pM NGF gave significantly less protection (Table 5, row 3).D3 alone afforded low but significant protection (Table 5, row 4), andprotection was enhanced with a combination of 10 pM NGF+10 μM D3 (Table5, row 6). The negative control C59 compound had no effect alone or inenhancing 10 pM NGF (Table 5, rows 5 and 7).

[0095] In other controls, neither D3 nor NGF protected B104 cells, wildtype HEK293 cells, or TrkB-expressing HEK293 cells from apoptosis. Hencethe trophic activity of NGF and D3 require TrkA expression, or at leastthe IgG-C2 domain of TrkA. Additionally, D3 did not enhance the trophiceffect of EGF suggesting that it may be NGF selective. Lastly, D3enhanced NGF protection of NIH3T3 cells stably transfected with TrkAcDNA but did not enhance NT-3 protection of NIH3T3 cells stablytransfected with trkC cDNA. These data indicate that D3 selectivelyaccentuates the trophic effect of NGF, and that expression of the p75low affinity NGF receptor is not required.

[0096] The proteolytic stability of D3 versus trypsin and papain wasassessed. D3 was first exposed to enzymatic treatment as describedpreviously (Saragovi et al., 1992), followed by gauging its biologicalactivity on 4-3.6 cells. Compound D3 remained fully active in trophicassays even after 1 hour of exposure to trypsin or pepsin, whereas NGFlost all activity within minutes under the same conditions.

[0097] D3 Induces Differentiation of Primary Cultures of Fetal DRG andFetal Septal Neurons

[0098] The effect of D3 on TrkA-mediated cellular differentiation wasassessed using two independent assays: morphometric analysis of DRGdissociated neurons and induction of ChAT activity in septal neuronalcultures. In the first of these assays, data indicate that DRG neuronalcultures undergo neurite outgrowth in response to D3, and that D3potentiates the effect of NGF (FIG. 1). In the second assay, ChATactivity was found to increase in response to NGF (Table 6, rows 1 and2) and to D3 alone (Table 6, rows 3-5), whereas C59 control had noeffect (Table 6, row 6). Increases in ChAT activity in response to 2 μMD3 alone were comparable to 10 pM exogenous NGF. Moreover, combinationsof 2 μM D3+10 pM NGF markedly increased ChAT activity, and were moreeffective than 400 pM NGF (Table 6, rows 8-10).

[0099] D3 Enhances or Stabilizes Putative TrkA•TrkA Homodimers.

[0100] Based on the data above, it was expected that D3 would induce orstabilize TrkA•TrkA interactions. This hypothesis was studiedbiochemically in 4-3.6 cells exposed to ligands, followed by cellsurface chemical cross-linking (FIG. 2).

[0101] The expected doublet consistent with previously reported TrkAmonomers of p110 and p140 were seen in all samples (FIG. 2, thickarrow). Bands of ˜300 kDa, consistent with the molecular weight ofTrkA•TrkA homodimers (FIG. 2, thin arrow), were seen in samples fromcells treated with TrkA ligands 1 nM NGF, 10 pM NGF, or 10 pM NGF+10 μMD3, and was also detected (albeit very more weakly) in cells treatedwith 10 μM D3 alone. The intensity of the band M_(r) 300 kDa, presumedto be TrkA dimers, was analyzed densitometrically from 4 independentexperiments standardized to 1 nM NGF (100%). There was a consistentincrease in dimers after treatment with D3 alone (21±4%) or 10 pM NGFalone (52±6%), which was higher after treatment with 10 pM NGF+10 μM D3(77±7%). Control cells cross-linked in the absence of ligand or cellsexposed to ligand but not-cross-linked did not have putative dimers.

[0102] TrkA homodimers are stable to SDS denaturation because ofcovalent cross-linking. Given that the efficiency of chemicalcross-linking is ˜1-4% of the total TrkA pool further biochemicalcharacterization of the complexes was precluded, other than the factthat they contain TrkA. The complexes may contain cross-linked NGF.However, it is unlikely that the bands comprise p75 becauseimmunoprecipitations with anti-p75 antibodies did not reveal anymaterial in the M_(r) of TrkA homodimers. Furthermore, material of M_(r)215 kDa that would comprise p75-TrkA heterodimers was not seenconsistently.

[0103] Biological Activity of Examples of Preferred Embodiments.

[0104] Examples of some preferred embodiments that mimic NGF-likeneurotrophic activity (Table 7) are agents termed D3, D53b-d, D21, P23,and P58. These agents, tested at 10 I, afford significant survival tocells expressing TrkA, but not to cells that do not express neurotrophinreceptors. Additionally, these agents synergize with suboptimalconcentrations of NGF (10 pM). NGF at 10 pM affords 32±6% survivalcompared to 1 nM NGF which in TrkA cells is standardized to 100%survival. NGF at 10 pM plus the indicated embodiments significantlyenhances cell survival.

[0105] Examples of some preferred embodiments that mimic NT-3-likeneurotrophic activity (Table 8) are agents termed P27 and P23. Theseagents, tested at 10 μM, afford significant survival to cells expressingTrkC, but not to cells that do not express neurotrophin receptors.Additionally, these agents synergize with suboptimal concentrations ofNT-3 (10 pM). NT-3 at 10 pM affords 29±1% survival compared to 1 nM NT-3which in TrkC cells is standardized to 100% survival. NT-3 at 10 pM plusthe indicated embodiments significantly enhances cell survival. Notethat P23 enhances survival of cells expressing TrkA and TrkC, hence itbehaves as NT-3 which is a ligand of both receptors.

[0106] Examples of some preferred embodiments that antagonize NGFneurotrophic activity (Table 9) are agents termed P42 and P43. Whilethese embodiments alone do not affect cell survival (Table 9, rows 4 and5), they do reduce the survival afforded by NGF (Table 9, rows 6-9). NGFat 1 nM (Table 9, row 2) affords 100% survival and this effect isreduced by P42 and by P43 respectively to 68% and 55% survival. Hencethese embodiments are antagonistic to NGF neurotrophic activity.

Discussion

[0107] A proteolytically stable β-turn peptidomimetic small moleculeagonist of the TrkA neurotrophin receptor. We showed that D3 binds TrkA,competes the binding of the TrkA agonist mAb 5C3, selectivelypotentiates trophic protection of TrkA-expressing cell lines andneuronal primary cultures, and induces the differentiation of primaryneuronal cultures. These results indicate that a small β-turnpeptidomimetic can activate a tyrosine kinase neurotrophin receptor thatnormally binds a relatively large protein ligand.

[0108] Recent advances in ligand mimicry have resulted from screeninglarge phage or peptide libraries, natural products, or chemicallibraries. However, most of the ligands described are antagonists, orotherwise require the dimerization of relatively large peptides, have a2-fold axis of symmetry that resemble a dimer, or are poorly soluble inphysiological buffers. In contrast, D3 is a small, non-symmetrical,proteolytically stable, highly water soluble peptidomimetic that bindsthe extracellular domain of TrkA.

[0109] Binding and ligand competition studies demonstrate selectiveinteraction of D3 with the extracellular domain of TrkA, rather than thecatalytic domain. Hence, the water solubility and extracellulartargeting of D3 mean that toxic organic solvents are not required topermeate the cell membrane.

[0110] What is the Role of pM Concentrations of NGF?

[0111] Given the low concentrations used in synergy with D3, it isunlikely that the effect of NGF was mediated by docking with the lowaffinity receptor p75. It is postulated that NGF acts by increasingTrkA•TrkA interactions whereas D3 stabilizes the homodimers or reducesthe rate of separation of receptor homodimers by inducing conformationalchanges.

[0112] In the present invention, the biological data shown are with lowμM concentrations of D3, which are optimal. As expected from theaffinity estimated for TrkA•D3 interactions, lower D3 concentrationsafford lower efficacy. It is noteworthy that while NGF•TrkA affinity is˜10⁻¹¹ M, optimal activity requires 2 nM NGF concentrations. Hence, D3is optimal at concentrations that approximate its K_(d) while NGF isoptimal at concentrations 100 fold over its K_(d). This difference isinterpreted to mean that D3 is more stable in solution, and this notionis supported by D3 resistance to proteolysis.

[0113] Ligand Binding Sites

[0114] D3 competitively blocks the binding of mAb 5C3 but it does notblock NGF. Moreover, the optimal agonistic activity of mAb 5C3 wasinhibited by D3 in a dose-dependent manner, while the agonistic effectof NGF was enhanced. It is unlikely that D3 does not block NGF becauseof affinity differences, because NGF•TrkA-ECD and 5C3•TrkA-ECDinteractions are both in the nM range.

[0115] Two factors could account for this result. First, both mAb 5C3and D3 dock onto a single and continuous epitope within the IgG-C2domain of TrkA, whereas NGF binds a discontinuous epitope within theIgG-C1 and IgG-C2 domains of TrkA (Perez et al., 1995), and otherdomains. This would facilitate mAb 5C3 blocking by D3 whereas NGF couldbind via its second docking site. Second, mAb 5C3 and NGF bind TrkA atsites partially overlapping but not identical (LeSauteur et al., 1996).Hence the data suggest that D3 binds TrkA at an epitope overlapping theagonistic mAb 5C3 “hot spot” of the IgG-C2 domain of TrkA, near the NGFdocking site. These observations may account for D3 synergizing with NGFand blocking mAb 5C3. The docking site is called “hot spot” because itdefines a functional site wherein ligands that bind the site can triggera function. That function may be (partial) agonistic or (partial)antagonistic.

[0116] The fact that D3 is bioactive and was selected from a relativelysmall pool of β-turn based compounds has broad implications for manyresearch initiatives involving protein-protein interactions.Particularly these notions can be applied to all members of theneurotrophin family of ligands and their receptors because they allfunction in a manner comparable to TrkA.NGF.

[0117] The present invention provides a small molecule peptidomimeticthat binds and activates TrkA. In the present invention it is found thata hybrid of a peptide and a small organic molecule designed to hold keyamino acid residues in a turn conformation within a small frameworkoffers a means to transform a peptide lead into an active organic smallmolecule. Hence, D3 represents the validation of the peptidomimeticconcept for the Trk family of tyrosine kinase receptors. This smallmolecule peptidomimetic ligand of TrkA that has neurotrophic activitymay be used to address neurodegenerative disorders, pain, neoplasias,and other pathologies (reviewed in (Saragovi and Burgess, 1999)) whereTrk receptors play a role.

[0118] Table 1. D3 and D3-Biotin Bind TrkA.

[0119] Binding of biotin-D3 to B104 cells (p75+ TrkA−) or 43.6 cells(p75+ TrkA+) was quantitated by FACScan analysis. Ligands arecontrol-biotin (an inactive biotinylated peptide) (row 2), D3-biotin(row 3), or D3-biotin with a 10-fold molar excess of D3 (row 4). Allligands were followed with avidin-FITC as a fluorescent label. Datashown are mean channel fluorescence (MCF) of bell-shaped histograms,5,000 events acquired. MCF data±sem are averaged from 3 independentexperiments. TABLE 1 D3 and D3-biotin bind TrkA. Binding of biotin-D3 toB104 cells (p75+ TrkA-) or 4-3.6 cells (p75+ TrkA+) was quantitated byFACScan analysis. Ligands are control- biotin (an inactive biotinylatedpeptide) (row 2), D3-biotin (row 3), or D3-biotin with a 10-fold molarexcess of D3 (row 4). All ligands were followed with avidin-FITC as aflourescent label. Data shown are mean channel flourescence (MCF) ofbell-shaped histograms, 5,000 events acquired. MCF data ± sem areaveraged from 3 independent experiments. MCF Ligand B104 4-3.6 untreated10 ± 3 13 ± 2 control-biotin 20 μM 11 ± 1 10 ± 3 D3-biotin 20 μM 10 ± 453 ± 4 D3-bio 20 μM + D3 200 μM 11 ± 2 17 ± 7

[0120] TABLE 2 D3 specifically blocks mAb 5C3 binding to cell surfaceTrkA. 4-3.6 cells were analyzed by FACScan for binding of anti-TrkA mAb5C3 or anti-p75 mAb MC192. Cells exposed to control primary mouse IgGwith or without 40 μM D3 afford identical background staining. For eachcondition 5,000 cells were acquired. Percentage maximal bindings werecalculated from the MCF of bell-shaped histograms, using the formula(TEST_(MCF)-background_(MCF))*100/ MAXIMAL_(MCF)-background_(MCF)). MCF± sem are averaged from 3 independent experiments. MAb (1 nM) CompetitorDose (μM) % Maximal binding 1 5C3 none 0 100 ± 0  2 5C3 D3 0.20 95 ± 4 35C3 D3 1 80 ± 3 4 5C3 D3 5 53 ± 5 5 5C3 D3 40 33 ± 4 6 5C3 C59 control40 97 ± 6 7 MC192 none 0 100 ± 0  8 MC192 D3 40 101 ± 2 

[0121] TABLE 3 D3 inhibits 5C3 · TrkA interactions in vitro. The bindingof mAb 5C3 (at constant 2 nM) to purified TrkA-ECD immobilized ontoELISA plates was measured in the absence or presence of competitors.Background (<2%) was the optical density of wells with all reactantsexcept immobilized TrkA-ECD. Data are averaged from 3 experiments, eachexperiment n = 4. Competitor Concentration % Binding ± added (μM) sem 1— — 100 ± 3  2 D3 0.05 100 ± 14 3 D3 0.2 89 ± 8 4 D3 1  64 ± 10 5 D3 5 43 ± 12 6 D3 20 38 ± 7 7 D3 40 31 ± 4 8 C59 40 96 ± 9

[0122] TABLE 4 D3 protects TrkA-expressing primary neurons fromapoptosis. and potentiates NGF. NGF-dependent primary neuronal culturesfrom embryonic rat DRGs were treated with the indicated ligands for atotal of 8 days. Cell survival was measured by MTT assays. Protectionwas calculated relative to optimal NGF (1 nM, 100% protection) withsubtraction of the O.D. of untreated cells. Shown is the O.D. from oneexperiment, mean ± sem, n = 4. % protection was averaged from 3experiments. Treatment Optical Density % Protection 1 untreated 256 ± 15 0 ± 2 2 NGF 1 nM 823 ± 28 100 ± 4  3 NGF 20 pM 316 ± 11  9 ± 1 4 NGF500 pM 535 ± 19 68 ± 3 5 D3 10 μM 405 ± 22 38 ± 2 6 Control C59 10 μM271 ± 8   0 ± 1 7 D3 10 μM + NGF 20 pM 471 ± 28 48 ± 3 8 D3 10 μM + NGF500 pM 603 ± 26 84 ± 3 9 D3 10 μM + NGF 1 nM 977 ± 38 120 ± 7 

[0123] TABLE 5 D3 potentiates NGF in protecting TrkA-expressing celllines from apoptosis by binding to the IgC2 domain of the receptor.4-3.6 cells or HEK 293 cells expressing TrkB/TrkA IgG-C2 chimericreceptor were treated with the indicated ligands for a total of 72hours. Survival was measured by MTT assays. % Protection was calculatedas in Table 4. Shown is the O.D. from one experiment, mean ± sem, n = 4.Percent protection was averaged from 6 (4-3.6 cells) or 3 (293-IgG-C2chimera) independent experiments. HEK 293-TrkB/ 4-3.6 cells TrkA chimeraOptical Optical Treatment Density % Protection Density % Protection 1untreated 64 ± 7  0 ± 2 32 ± 5 0 ± 4 2 1 nM NGF 412 ± 24 100 ± 6  350 ±12 100 ± 4  3 10 pM NGF 205 ± 19 40 ± 5 88 ± 8 18 ± 5  4 10 μM D3 95 ± 9 8 ± 2 69 ± 7 9 ± 3 5 10 μM C59 76 ± 4  2 ± 1 30 ± 7 −1 ± 2   6 10 μMD3 + 255 ± 14 55 ± 3 165 ± 11 42 ± 5  10 pM NGF 7 10 μM C59 + 209 ± 1741 ± 4 90 ± 9 21 ± 6  10 pM NGF

[0124] TABLE 6 D3 induces ChAT synthesis. Septal neuronal cultures weretreated as indicated for a total of 8 days. ChAT activity (pmolAch/min/well ± sem) was measured at day 8. Average ± sem. Data averagedfrom 3 independent experiments, each experiment n = 4. Treatment ChATActivity Fold Increase 1 10 pM NGF 0.42 ± 0.07 1.4 2 400 pM NGF 0.72 ±0.10 2.41 3 0.2 μM D3 0.37 ± 0.05 1.23 4 2 μM D3 0.44 ± 0.02 1.47 5 20μM D3 0.48 ± 0.06 1.56 6 20 μM C59 control 0.30 ± 0.05 1 7 untreated0.31 ± 0.07 1 8 0.2 μM D3 + 10 pM NGF 0.60 ± 0.04 2.00 9 2 μM D3 + 10 pMNGF 0.76 ± 0.03 2.53 10 20 μM D3 + 10 pM NGF 0.79 ± 0.04 2.63

[0125] TABLE 7 TrkA agonistic activity of examples of preferredembodiments NIH3T3 fibroblasts transfected with and expressing TrkA orTrkC receptors, or untransfected wild type controls (NIH wt), weretreated with the indicated ligands for a total of 72 hours.TrkA-expressing cells respond optimally to NGF. TrkC-expressing cellsrespond optimally to NT-3. Tests were done with agents alone or in thepresence of suboptimal concentrations of NGF (10 pM). Survival wasmeasured by MTT assays. % Protection was calculated as in Table 4. Shownis the O.D. from one experiment, mean ± sem, n = 4. Each experiment wasrepeated 3 times or more. NIH-TrkA NIH-TrkC NIH wt Treatment %Protection % Protection % Protection 1 untreated  0 ± 4 0 ± 2 0 ± 5 2 1nM NGF 100 ± 2  5 ± 6  5 ± 12 3 10 pM NGF 32 ± 6 2 ± 4 5 ± 7 6 10 μM D3 8 ± 4 3 ± 3 −3 ± 5   7 10 μM D3 + 10 pM NGF 51 ± 5 6 ± 2 0 ± 3 10 10 μMD53b-d 11 ± 4 4 ± 2 3 ± 5 11 10 μM D53b-d + 10 pM 45 ± 4 3 ± 1 −4 ± 6  NGF 12 10 μM D21 16 ± 3 6 ± 4 3 ± 2 13 10 μM D21 + 10 pM 60 ± 5 5 ± 1 3± 3 NGF 14 10 μM P23 15 ± 2 8 ± 2 0 ± 3 15 10 μM P23 + 10 pM 52 ± 3 11 ±5  1 ± 3 NGF 16 10 μM P58 21 ± 7 6 ± 5 −1 ± 3   17 10 μM P58 + 10 pM 43± 6 5 ± 3 4 ± 2 NGF

[0126] TABLE 8 TrkC agonistic activity of examples of preferredembodiments NIH3T3 fibroblasts transfected with and expressing TrkA orTrkC receptors, or untransfected wild type controls (NIH wt), weretreated with the indicated ligands for a total of 72 hours.TrkA-expressing cells respond optimally to NGF and to a lesser degree toNT-3. TrkC-expressing cells respond optimally to NT-3. Tests were donewith agents alone or in the presence of suboptimal concentrations ofNT-3 (10 pM). Survival was measured by MTT assays. % Protection wascalculated as in Table 4. Shown is the O.D. from one experiment, mean ±sem, n = 4. Each experiment was repeated 3 times or more. NIH-TrkANIH-TrkC NIH wt Treatment % Protection % Protection % Protection 1untreated 0 ± 4  0 ± 2 0 ± 5 2 1 nM NT-3 17 ± 3  100 ± 2  −2 ± 5   3 10pM NT-3 3 ± 2 29 ± 1 1 ± 4 4 10 μM P27 5 ± 2 13 ± 3 −2 ± 4   5 10 μMP27 + 10 pM 4 ± 1 62 ± 6 5 ± 4 NT-3 6 10 μM P23 15 ± 2   8 ± 2 0 ± 3 710 μM P23 + 10 pM 21 ± 3  51 ± 7 1 ± 3 NT-3

[0127] TABLE 9 TrkA antagonistic activity of examples of preferredembodiments NIH3T3 fibroblasts transfected with and expressing TrkA weretreated with the indicated ligands for a total of 72 hours.TrkA-expressing cells respond optimally to 1 nM NGF and suboptimally to10 nM NGF. Survival was measured by MTT assays. % Protection wascalculated as in Table 4. Shown is the O.D. from one experiment, mean ±sem, n = 4. Each experiment was repeated 3 times or more. Nd: not doneNIH-TrkA NIH wt Treatment % Protection % Protection 1 untreated 0 ± 5 0± 4 2 1 nM NGF 100 ± 4  4 ± 6 3 10 pM NGF 28 ± 5  3 ± 5 4 10 μM P42 5 ±2 0 ± 3 5 10 μM P43 4 ± 3 2 ± 4 6 1 nM NGF + 10 μM P42 68 ± 5  nd 7 10pM NGF + 10 μM P42 9 ± 4 nd 8 1 nM NGF + 10 μM P43 55 ± 7  nd 9 10 pMNGF + 10 μM P43 12 ± 3  nd

[0128] Further Considerations

[0129] The molecular nature of NT-3/TrkC interactions is important forthe following reasons. Many protein-protein interactions occur viacontact at a few key regions, “hot spots”, rather than extensiveinteractions over the whole protein surface. These generally involve10-30 contact side chains on discontinuous portions of each primarysequence. However, a relatively small fraction of these side chains arerequired for tight binding at the interfaces. Small molecules thatinteract with hot spots can interfere with the normal protein-proteininteractions making the concept of hormone mimicry viable.

[0130] Previous results provide evidence that the turn regions of theneurotrophin NGF are hot-spots for the NGF/TrkA interaction (LeSauteuret al, 1995). NGF is a 22 kDa protein, which exists and functions as adimer. It is highly conserved across species. Mature NT-3 shares 50identical amino acids with NGF, mostly focused in regions that promotethe common tertiary structure (eg all six Cys residues of the cysteineknot are conserved). The dimer interface region is composed of β-strandsthat maintains the conformation and disposition of structural motifs;these hydrophobic core residues are highly conserved amongst allneurotrophins. Conversely, the turn regions are highly variable, andappear to determine receptor-binding specificity.

[0131] The strong structural similarities between NGF and NT-3 (in theBDNF heterodimer) indicate the turn regions of NT-3 highlighted in FIG.4 are important in docking of NT-3 with TrkC. Additionally mimics of theturn regions of NT-3 may also bind TrkA and p75 (as NT-3 does)

[0132] The data from studies of chimeric proteins is as follows. OneNT-3 chimera that expresses NGF residues 1-66 and 115-122 (numberingaccording to NGF) exhibits gain of NGF-like function, with fullretention of NT-3 activity. These findings imply the sequences thatconfer NT-3 like properties are contained within the region thatcorresponds to NGF residues 67-114. Another NT-3 chimera studiedcontained the NGF sequences at the N-terminus and at β-loop 3 region (caresidues 91-98). This recombinant had enhanced NGF function anddiminished NT-3 activity relative to wild type NT-3. These data implythat a major contribution towards NT-3 binding and activity isattributable to β-loop 3. This is consistent with the studies of thefirst chimera because the β-loop 3 is within the region corresponding toresidues 67-114 that was found to contain key regions for binding.Another study of NT-3/NGF chimeric proteins found that β-loop 3 was acritical region for determining specificity, and that proximal Arg andTyr residues may enhance the binding.

[0133] Mutagenesis experiments reveal scattered residues of NT-3 thatcontribute to binding. Thus some loss of NT-3/TrkC affinity and loss ofactivity is seen upon substitution of six residues of NT-3 with thecorresponding residues in NGF (S73D, F86Y, K88R, F101W, A107S, andV111A). These amino acids are discontinuous in the primary sequence, butthey are proximal to β-loop 3 in the folded dimeric neurotrophin. Nextto the β-loop 3 of NT-3 there is an arginine that is conserved betweenNGF and NT-3 but seems to play a different role in each neurotrophin.Significant loss of NT-3 bioactivity was seen in an NT-3 R103A mutant;however, no loss of NGF bioactivity was observed in a NGF R103A mutant.This is indicative of differences in the way these neurotrophins bind totheir receptors. Moreover, adjacent to R103, there is a phenylalanine(F104) in NGF and a tryptophan (W104) in NT-3. These hydrophobicresidues are solvent exposed and their substitution also leads todecreased bioactivity, suggesting a role either in binding or instabilizing an active conformation.

[0134] It appears that the N-termini of the neurotrophins may play animportant role in binding to their receptors. This region is a moredifficult target for mimicry because the N-terminus of NT-3 (and that ofNGF) is “unstructured” in solution and in the solid state. Modelingindicates that the N-terminus is composed of two subdomains comprisingresidues 1-8 and residues 9-11. Residues 1-8 are flexible, but 9-11 arerigid and maintain an electrostatic interaction between E(11) andR(118). Consequently, a long fold locates residues 1-8 near -loop2/-loop 3.

[0135] NGF/p75 and NT-3/p75 interactions are at least partially mediatedby β-loop 1 of the neurotrophin which features positively charged aminoacids. They may also involve amino acids R114 and K115. Overall, theseresidues are discontinuous with the -loop 1 primary sequence, but arepacked closely to it in the 3-D structure.

[0136] On the basis of the data above, it is appropriate to specificallyincorporate one or more of the residues found in neurotrophins atpositions 1-11; 29-34; 42-48; and 91-98. These will be the primarytargets for lead discovery.

[0137] Table 10 provides sequence alignments. TABLE 10 Alignment ofamino acid sequences of mature neurotrophins in regions predicted toconvey receptor binding and specificity. region/domain N-terminus β-loop1 β-loop 2 NGF mouse S S T H P V F H T A T D I K G K E V T E V N I N N SV F rat S S T H P V F H T A T D I K G K E V T E V N I N N S V F human SS S H P I F H T A T D I K G K E V M E V N I N N S V F NT-3 rat Y A E H KS H S A I D I R G H Q V T E I K T G N S P V human Y A E H K S H S A I DI R G H Q V T E I K T G N S P V BDNF human H S D P A R R G E T A V D M SG G T H S E K V P V S K G Q region/domain β-loop 3 GF mouse R Q L T T DE K Q A A W R F rat K Q L T T D D K Q A A W R F human K Q L T M D G K QA A W R F NT-3 rat K Q L T S E N N K L V G W R W human K Q L T S E N N KL V G W R W BDNF human R A L T M D S K K R I G W R F

[0138] Abbreviations BDNF, Brain Derived Neurotrophic Factor BOC,tert-butoxycarbonyl ChAT, Choline Acetyl Transferase DMF,dimethylformamide DRG: dorsal root ganglia ELISA: enzyme-linkedimmunosorbent assay FACScan, Fluorescent Activated Cell Scanner FITC,fluorescein isothiocyanate FMOC, fluorenyloxycarbonyl MCF, mean channelfluorescence MTT, 3(4,5-Dimethylthiazolyl-2)-2,5-diphenyl tetrazoliumbromide NGF, nerve growth factor NT-3, neurotrophin-3 RIA:radioimmunoassay TFA, trifluoroacetic acid Trt, trityl

REFERENCES

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[0140] Feng, Y, Wang, Z, Jin, S, and Burgess, K (1998) SNAr CyclizationsTo Form Cyclic Peptidomimetics of beta-turns. J. Am. Chem. Soc.120:10768-9.

[0141] LeSauteur, L, Maliartchouk, S, Jeune, H L, Quirion, R, andSaragovi, H U (1996) Potent Human p140-TrkA Agonists Derived from anAnti-receptor Antibody. J. Neurosci. 16:1308-16.

[0142] LeSauteur, L, Wei, L, Gibbs, B, and Saragovi, H U (1995) SmallPeptide Mimics of Nerve Growth Factor Bind TrkA Receptors and AffectBiological Responses. J. Biol. Chem. 270:6564-9.

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1. A neurotrophin receptor agonist or antagonist pharmaceuticalcomposition comprising an acceptable neurotrophin receptor agonisticamount of a neutrophin mimicking β-turn peptidomimetic cyclic compound,in association with a pharmaceutically acceptable carrier.
 2. Acomposition according to claim 1, wherein said compound has amacrocyclic ring of 13 to 17 ring atoms.
 3. A composition according toclaim 1 or 2, wherein said compound has one or more side chains on saidmacrocyclic ring, which one or more side chains extend from backbonering atoms.
 4. A composition according to claim 3, wherein said one ormore, side chains correspond to residues found within β-turns of aneurotrophin.
 5. A composition according to claim 4, wherein saidneurotrophin is nerve growth factor (NGF), neurotrophin-3 (NT-3),neurotrophin-4/5 (NT4/5), or brain derived neurotrophic factor (BDNF).6. A composition according to claim 4, wherein said neurotrophin bindsto a receptor.
 7. A composition according to claim 4, wherein saidneurotrophin receptor is TrkA, TrkB, TrkC or p75.
 8. A compositionaccording to claim 1, wherein said cyclic compound is a β-turnpeptidomimetric cyclic compound of formula (I)

wherein R¹ and R³ are selected from alkyl or aryl substituents found ina natural or unnatural amino acid; R² and R⁴ are hydrogen or alkyl; R⁵and R⁶ are hydrogen; or R¹ and R² or R³ and R⁴ can together with thecarbon atom to which they are attached form a cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl group, and Z, Y, X, LINKER and n are asdefined herein.
 9. A composition according to claim 1, wherein saidcompound is selected from


10. A method of treating or preventing a neurotrophin receptor mediateddisorder in a patient comprising administering to a patient in need, anacceptable neurotrophin receptor agonistic or antagonistic amount of aneurotrophin mimicking β-turn peptidomimetic cyclic compound.
 11. Use ofβ-turn peptidomimetic cyclic compounds in evaluating structuralrequirements of neutrophin mimicking β-turn peptidomimetic cycliccompounds.
 12. Use of a neurotrophin mimicking β-turn peptidomimeticcyclic compound in the manufacture of a medicament for treating orpreventing a neurotrophin receptor mediated disorder.
 13. Use of claim12, wherein said compound is selected from


14. A β-turn peptidomimetic cyclic compound of formula (I)

wherein R¹ and R³ are selected from alkyl or aryl substituents found ina natural or unnatural amino acid; R² and R⁴ are hydrogen or alkyl; R⁵and R⁶ are hydrogen; or R¹ and R² or R³ and R⁴ can together with thecarbon atom to which they are attached form a cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl group, and Z, Y, X, LINKER and n are asdefined herein.
 15. A cyclic compound of claim 14, in which themacrocyclic ring containing X has 13 to 17 ring atoms.
 16. A cycliccompound of claim 15, having one or more side chains on saidmacrocyclic-ring, which one or more side chains extend from backbonering atoms.
 17. A compound of claim 14, 15 or 16, wherein R¹ and R³ arederived from a sequence of different amino acids side chains selectedfrom natural and synthetic amino acids.
 18. A compound of claim 14, 15,16 or 17, wherein X is —O—, —S— or —NH—.
 19. A compound of claim 14formula


20. A compound of claim 14 formula:


21. A compound of claim 14, selected from


22. A method of screening and/or evaluating necessary structuralrequirements of agonists and antagonists for neurotrophin receptorswhich exploits a cyclic compound as defined in any one of claims 14 to21 in competition with a test compound under investigation.
 23. Use of acompound as defined in any one of claims 14 to 21 for identifyingfunctionally important receptor domains, in binding assays.